ELECTROPHORESIS OF PROTEINS AND PROTEIN-PROTEIN COMPLEXES IN NATIVE POLYACRYLAMIDE GELS USING A HORIZONTAL GEL APPARATUS

Electrophoresis of proteins and protein-protein complexes in polyacryl amide gels under native conditions using a horizontal gel apparatus is described. The advantage of this system is that it permits the detect ion of both negatively and positively charged proteins as well as prot ein-protein complexes in the same gel. During electrophoresis, a conti nuous gel sandwiched between two glass plates is placed horizontally o n the platform and submerged in a reservoir buffer. The sample wells a re made along the center of the gel, allowing positively and negativel y charged proteins to migrate toward the cathode and anode, respective ly. Several proteins with varying molecular weights and isoelectric po int (pI) values and pairs of proteins capable of forming protein-prote in complexes were chosen as model systems to illustrate the methodolog y. The effects of several parameters on the performance of the gel sys tem including protein molecular weight, pI, and gel concentration were also examined and the results obtained by this method are comparable to those obtained by the vertical system. Following electrophoresis, b oth negatively and positively charged proteins as well as protein-prot ein complexes can be transferred by electroblotting onto polyvinyliden e difluoride membranes for further analyses. (C) 1994. Academie Press, Inc.